Mitochondrial DNA in forensic analyses

  • Irena Zupanič Pajnič
Keywords: skeletal remains, nails, hair, feces, urine


This review article presents mitochondrial DNA (mtDNA) analyses in forensic genetics. Typing of nuclear DNA in old and poorly preserved biological samples is often unsuccessful and instead of nuclear DNA, mtDNA polymorphisms can be used for human identification. MtDNA is due to multy copy nature and circular conformation less prone to degradation and retain longer time. The most polymorphic control region is regularly used in forensic casework and mtDNA heteroplasmy must be considered in the identification cases and analyses of biological traces. The poorly preserved biological samples are mainly old skeletal remains - bones and teeth, old nails, feces, urine and hair shafts. The latter are micro traces, often found by criminalists at crime scenes. We will describe each type of biological material in more detail. In addition to morphological structure, we will pay special attention to the environmental impacts on the preservation of DNA in each type of biological material, optimization of extraction methods for effective isolation and optimal sampling. Due to low amounts of DNA, these samples are exposed to DNA contamination with persons involved in sampling procedures and genetic testing. To prevent and track potential contamination, different measures are used and they will be described. MtDNA analyses are often used in forensics to solve different cases and some applications of mtDNA in Slovenia and worldwide will be discussed.


Download data is not yet available.


Zupanič Pajnič I, Šterlinko H, Bala ic J, Komel R. Parentage testing with 14 STR loci and population data for 5 STRs in the Slovenian population. Int J Legal Med. 2001;114(3):178-80.

Zupanič Pajnič I, Balažic J, Komel R. Sequence polymorphism of the mitochondrial DNA control region in the Slovenian population. Int J Legal Med. 2004;118(1):1-4.

Fisher DL, Holland MM, Mitchell L, Sledzik PS, Wilcox AW, Wadhams M, et al. Extraction, evaluation, and amplification of DNA from decalcified and undecalcified United States Civil War bone. J Forensic Sci. 1993;38(1):60-8.

Coble MD, Vallone PM, Just RS, Diegoli TM, Smith BC, Parsons TJ. Effective strategies for forensic analysis in the mitochondrial DNA coding region. Int J Legal Med. 2006;120(1):27-32.

Zupanič Pajnič I. Identifikacija oseb iz starih in slabo ohranjenih bioloških materialov s polimorfizmi mitohondrijske DNA. [PhD Thesis]. Ljubljana: I. Zupanič Pajnič; 2007.

Obal M. Uporaba različnih skeletnih elementov za genetsko identifikacijo žrtev druge svetovne vojne. [Master's Thesis]. Ljubljana: M. Obal; 2018.

Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290(5806):457-65.

Vogel F, Motulsky AG. Human genetics - problems and approaches. Berlin: Springer-Verlag; 2018. 1997. pp. 83-7, 114-27, 145-93, 495-583, 593-54, 610-21, 705-11.

Bendall KE, Macaulay VA, Sykes BC. Variable levels of a heteroplasmic point mutation in individual hair roots. Am J Hum Genet. 1997;61(6):1303-8.

Howell N, Kubacka I, Mackey DA. How rapidly does the human mitochondrial genome evolve? Am J Hum Genet. 1996;59(3):501-9.

Holland MM, Fisher DL, Mitchell LG, Rodriquez WC, Canik JJ, Merril CR, et al. Mitochondrial DNA sequence analysis of human skeletal remains: identification of remains from the Vietnam War. J Forensic Sci. 1993;38(3):542-53.

Hopwood AJ, Mannucci A, Sullivan KM. DNA typing from human faeces. Int J Legal Med. 1996;108(5):237-43.

Tully G, Bär W, Brinkmann B, Carracedo A, Gill P, Morling N, et al. Considerations by the European DNA profiling (EDNAP) group on the working practices, nomenclature and interpretation of mitochondrial DNA profiles. Forensic Sci Int. 2001;124(1):83-91.

Sullivan KM, Hopgood R, Gill P. Identification of human remains by amplification and automated sequencing of mitochondrial DNA. Int J Legal Med. 1992;105(2):83-6.

Piercy R, Sullivan KM, Benson N, Gill P. The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int J Legal Med. 1993;106(2):85-90.

Lutz S, Wittig H, Weisser HJ, Heizmann J, Junge A, Dimo-Simonin N, et al. Is it possible to differentiate mtDNA by means of HVIII in samples that cannot be distinguished by sequencing the HVI and HVII regions? Forensic Sci Int. 2000;113(1-3):97-101.

Cann RL, Stoneking M, Wilson AC. Mitochondrial DNA and human evolution. Nature. 1987;325(6099):31-6.

Lorente JA, Entrala C, Alvarez JC, Lorente M, Arce B, Heinrich B, et al. Social benefits of non-criminal genetic databases: missing persons and human remains identification. Int J Legal Med. 2002;116(3):187-90.

Bär W, Brinkmann B, Budowle B, Carracedo A, Gill P, Holland M, et al. DNA commission of the International society for forensic genetics: guidelines for mitochondrial DNA typing. Int J Legal Med. 2000;113(4):193-6.

Allen M, Engström AS, Meyers S, Handt O, Saldeen T, von Haeseler A, et al. Mitochondrial DNA sequencing of shed hairs and saliva on robbery caps: sensitivity and matching probabilities. J Forensic Sci. 1998;43(3):453-64.

Parson W, Brandstätter A, Alonso A, Brandt N, Brinkmann B, Carracedo A, et al. The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives. Forensic Sci Int. 2004;139(2-3):215-26.

Linch CA, Whiting DA, Holland MM. Human hair histogenesis for the mitochondrial DNA forensic scientist. J Forensic Sci. 2001;46(4):844-53.

Wilson MR, Polanskey D, Replogle J, DiZinno JA, Budowle B. A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes. Hum Genet. 1997;100(2):167-71.

Tully G, Barritt SM, Bender K, Brignon E, Capelli C, Dimo-Simonin N, et al. Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts. Forensic Sci Int. 2004;140(1):1-11.

Calloway CD, Reynolds RL, Herrin GL, Anderson WW. The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age. Am J Hum Genet. 2000;66(4):1384-97.

Gill P, Ivanov PL, Kimpton C, Piercy R, Benson N, Tully G, et al. Identification of the remains of the Romanov family by DNA analysis. Nat Genet. 1994;6(2):130-5.

Ivanov PL, Wadhams MJ, Roby RK, Holland MM, Weedn VW, Parsons TJ. Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nat Genet. 1996;12(4):417-20.

Parsons TJ, Muniec DS, Sullivan K, Woodyatt N, Alliston-Greiner R, Wilson MR, et al. A high observed substitution rate in the human mitochondrial DNA control region. Nat Genet. 1997;15(4):363-8.

Bendall KE, Macaulay VA, Baker JR, Sykes BC. Heteroplasmic point mutations in the human mtDNA control region. Am J Hum Genet. 1996;59(6):1276-87.

Campos PF, Craig OE, Turner-Walker G, Peacock E, Willerslev E, Gilbert MT. DNA in ancient bone - where is it located and how should we extract it? Ann Anat. 2012;194(1):7-16.

Allen C, Harper V. Laboratory Manual for Anatomy and Physiology. 4th ed.. Hoboken (NJ): Wiley; 2011. pp. 96-8.

Kemp BM, Smith DG. Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int. 2005;154(1):53-61.

Hochmeister MN, Budowle B, Borer UV, Eggmann U, Comey CT, Dirnhofer R. Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains. J Forensic Sci. 1991;36(6):1649-61.

Hagelberg E, Sykes B, Hedges R. Ancient bone DNA amplified. Nature. 1989;342(6249):485.

Miloš A, Selmanović A, Smajlović L, Huel RL, Katzmarzyk C, Rizvić A, et al. Success rates of nuclear short tandem repeat typing from different skeletal elements. Croat Med J. 2007;48(4):486-93.

Hansen HB, Damgaard PB, Margaryan A, Stenderup J, Lynnerup N, Willerslev E, et al. Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum. PLoS One. 2017;12(1):e0170940.

Siriboonpiputtana T, Rinthachai T, Shotivaranon J, Peonim V, Rerkamnuaychoke B. Forensic genetic analysis of bone remain samples. Forensic Sci Int. 2018;284:167-75.

Mundorff A, Davoren JM. Examination of DNA yield rates for different skeletal elements at increasing post mortem intervals. Forensic Sci Int Genet. 2014;8(1):55-63.

Andronowski JM, Mundorff AZ, Pratt IV, Davoren JM, Cooper DM. Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach. Forensic Sci Int Genet. 2017;28:211-8.

Alvarez García A, Muñoz I, Pestoni C, Lareu MV, Rodríguez-Calvo MS, Carracedo A. Effect of environmental factors on PCR-DNA analysis from dental pulp. Int J Legal Med. 1996;109(3):125-9.

Rubio L, Martinez LJ, Martinez E, Martin de las Heras S. Study of short- and long-term storage of teeth and its influence on DNA. J Forensic Sci. 2009;54(6):1411-3.

Higgins D, Austin JJ. Teeth as a source of DNA for forensic identification of human remains: a review. Sci Justice. 2013;53(4):433-41.

Mansour H, Krebs O, Sperhake JP, Augustin C, Koehne T, Amling M, et al. Cementum as a source of DNA in challenging forensic cases. J Forensic Leg Med. 2018;54:76-81.

Smith BC, Fisher DL, Weedn VW, Warnock GR, Holland MM. A systematic approach to the sampling of dental DNA. J Forensic Sci. 1993;38(5):1194-209.

Higgins D, Kaidonis J, Austin J, Townsend G, James H, Hughes T. Dentine and cementum as sources of nuclear DNA for use in human identification. Aust J Forensic Sci. 2011;43(4):287-95.

Petrovič D, Zorc M. Histologija. Ljubljana: Univerza v Ljubljani, Medicinska fakulteta, Inštitut za histologijo in embriologijo; 2005. pp. 35-42, 115-24, 166-68.

Gilbert MT, Janaway RC, Tobin DJ, Cooper A, Wilson AS. Histological correlates of post mortem mitochondrial DNA damage in degraded hair. Forensic Sci Int. 2006;156(2-3):201-7.

Schlenker A, Grimble K, Azim A, Owen R, Hartman D. Toenails as an alternative source material for the extraction of DNA from decomposed human remains. Forensic Sci Int. 2016;258:1-10.

Cline RE, Laurent NM, Foran DR. The fingernails of Mary Sullivan: developing reliable methods for selectively isolating endogenous and exogenous DNA from evidence. J Forensic Sci. 2003;48(2):328-33.

Hellmann A, Rohleder U, Schmitter H, Wittig M. STR typing of human telogen hairs—a new approach. Int J Legal Med. 2001;114(4-5):269-73.

Gallimore JM, McElhoe JA, Holland MM. Assessing heteroplasmic variant drift in the mtDNA control region of human hairs using an MPS approach. Forensic Sci Int Genet. 2018;32:7-17.

Linch CA, Smith SL, Prahlow JA. Evaluation of the human hair root for DNA typing subsequent to microscopic comparison. J Forensic Sci. 1998;43(2):305-14.

Graffy EA, Foran DR. A simplified method for mitochondrial DNA extraction from head hair shafts. J Forensic Sci. 2005;50(5):1119-22.

Baker LE, McCormick WF, Matteson KJ. A silica-based mitochondrial DNA extraction method applied to forensic hair shafts and teeth. J Forensic Sci. 2001;46(1):126-30.

McNevin D, Wilson-Wilde L, Robertson J, Kyd J, Lennard C. Short tandem repeat (STR) genotyping of keratinised hair. Part 2. An optimised genomic DNA extraction procedure reveals donor dependence of STR profiles. Forensic Sci Int. 2005;153(2-3):247-59.

Johnson DJ, Martin LR, Roberts KA. STR-typing of human DNA from human fecal matter using the QIAGEN QIAamp stool mini kit. J Forensic Sci. 2005;50(4):802-8.

Roy R. Analysis of human fecal material for autosomal and Y chromosome STRs. J Forensic Sci. 2003;48(5):1035-40.

Vandenberg N, van Oorschot RA. Extraction of human nuclear DNA from feces samples using the QIAamp DNA Stool Mini Kit. J Forensic Sci. 2002;47(5):993-5.

Sípoli Marques MA, Pinto Damasceno LM, Gualberto Pereira HM, Caldeira CM, Pereira Dias BF, de Giacomo Vargens D, et al. DNA typing: an accessory evidence in doping control. J Forensic Sci. 2005;50(3):587-92.

Tsongalis GJ, Anamani DE, Wu AH. Identification of urine specimen donors by the PM+DQA1 amplification and typing kit. J Forensic Sci. 1996;41(6):1031-4.

Linfert DR, Wu AH, Tsongalis GJ. The effect of pathologic substances and adulterants on the DNA typing of urine. J Forensic Sci. 1998;43(5):1041-5.

Milde A, Haas-Rochholz H, Kaatsch HJ. Improved DNA typing of human urine by adding EDTA. Int J Legal Med. 1999;112(3):209-10.

Tsuchimochi T, Iwasa M, Maeno Y, Koyama H, Inoue H, Isobe I, et al. Chelating resin-based extraction of DNA from dental pulp and sex determination from incinerated teeth with Y-chromosomal alphoid repeat and short tandem repeats. Am J Forensic Med Pathol. 2002;23(3):268-71.

Rohland N, Hofreiter M. Ancient DNA extraction from bones and teeth. Nat Protoc. 2007;2(7):1756-62.

Parson W, Gusmão L, Hares DR, Irwin JA, Mayr WR, Morling N, et al. DNA Commission ofthe International Society for Forensic Genetics: revised and extended guidelines for mtDNAtyping. Forensic Sci Int Genet. 2014;13:134-42.

Zupanič Pajnič I. Extraction of DNA from Human Skeletal Material. In: Goodwin W. Forensic DNA Typing Protocols. New York: Humana Press; 2016. pp. 89-108.

Jehaes E, Decorte R, Peneau A, Petrie JH, Boiry PA, Gilissen A, et al. Mitochondrial DNA analysis on remains of a putative son of Louis XVI, King of France and Marie-Antoinette. Eur J Hum Genet. 1998;6(4):383-95.

Anslinger K, Weichhold G, Keil W, Bayer B, Eisenmenger W. Identification of the skeletal remains of Martin Bormann by mtDNA analysis. Int J Legal Med. 2001;114(3):194-6.

Parson W, Brandstätter A, Niederstätter H, Grubwieser P, Scheithauer R. Unravelling the mystery of Nanga Parbat. Int J Legal Med. 2007;121(4):309-10.

Lleonart R, Riego E, Saínz de la Peña MV, Bacallao K, Amaro F, Santiesteban M, et al. Forensic identification of skeletal remains from members of Ernesto Che Guevara’s guerrillas in Bolivia based on DNA typing. Int J Legal Med. 2000;113(2):98-101.

Mukaida M, Kimura H, Takada Y, Masuda T, Nakata Y. The personal identification of many samples recovered from under the sea. Forensic Sci Int. 2000;113(1-3):79-85.

Handt O, Richards M, Trommsdorff M, Kilger C, Simanainen J, Georgiev O, et al. Molecular genetic analyses of the Tyrolean Ice Man. Science. 1994;264(5166):1775-8.

Krings M, Stone A, Schmitz RW, Krainitzki H, Stoneking M, Pääbo S. Neandertal DNA sequences and the origin of modern humans. Cell. 1997;90(1):19-30.

Andelinović S, Sutlović D, Erceg Ivkosić I, Škaro V, Ivkosić A, Paić F, et al. Twelve-year experience in identification of skeletal remains from mass graves. Croat Med J. 2005;46(4):530-9.

Parsons TJ, Huel RM, Bajunović Z, Rizvić A. Large scale DNA identification: the ICMP experience. Forensic Sci Int Genet. 2019;38:236-44.

Zupanič Pajnič I, Gornjak Pogorelc B, Balažic J. Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia. Int J Legal Med. 2010;124(4):307-17.

Zupanič Pajnič I. Molecular genetic identification of the Slovene home guard victims. Zdrav Vestn. 2008;77:745-50.

Zupanič Pajnič I, Petaros A, Balažic J, Geršak K. Searching for the mother missed since the Second World War. J Forensic Leg Med. 2016;44:138-42.

Strobl C, Eduardoff M, Bus MM, Allen M, Parson W. Evaluation of the precision ID whole MtDNA genome panel for forensic analyses. Forensic Sci Int Genet. 2018;35:21-5.

Sturk-Andreaggi K, Peck MA, Boysen C, Dekker P, McMahon TP, Marshall CK. AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data. Forensic Sci Int Genet. 2017;31:189-97.

How to Cite
Zupanič Pajnič I. Mitochondrial DNA in forensic analyses. ZdravVestn [Internet]. 24Nov.2019 [cited 6Apr.2020];89(1-2):55-2. Available from:

Most read articles by the same author(s)